Modulation of host cell innate immune proteins by viral non-coding RNAs Open Access
Vachon, Virginia Katherine (2015)
Published
Abstract
Precise control of protein synthesis is essential for regulation of normal cellular processes and responses to cell stress. In addition, the innate immune response exploits varied strategies of translational control in order to limit viral protein synthesis and thus replication. The double-stranded RNA-activated protein kinase (PKR) and 2'-5'-oligoadenylate synthetases (OAS) sense cytosolic double-stranded RNA, a molecular hallmark of virus infection, to initiate general shut down of protein synthesis. The central importance of PKR and OAS is highlighted by numerous diverse strategies employed by a range of viruses to thwart their effects. Among them, the Adenovirus non-coding (nc)RNA transcript, VA RNAI, accumulates to high levels in later stages of infection, and is critical for efficient adenoviral replication. VA RNAI is well recognized for its inhibition of dsRNA-activated protein kinase (PKR)-mediated shut-down of general translation but, counterintuitively, VA RNAI activates rather than inhibits OAS1. The fundamental basis for control of these proteins by VA RNAI and other viral or cellular non-coding RNAs, including the range of RNA sequences and structures capable of regulating their activity remains underexplored. Here, VA RNAI is used as a model to further our understanding of RNA-mediated regulation of PKR and OAS1. Following a review of the current state of knowledge surrounding the multifaceted roles of VA RNAI, a description is provided of methods developed for solution-based probing of VA RNAI and other short RNA structures. These methods are then applied to a study on the role of VA RNAI structure in PKR inhibition. Finally, using VA RNAI as a starting point, discovery and characterization of a general, novel motif for optimal activation of OAS1 is presented. Defining the molecular signatures which govern the activities of PKR and OAS1 is essential to understanding how these proteins maximize their protective role against a broad range of pathogens while accurately discriminating between foreign and self molecules, avoiding inadvertent activation. Detailed knowledge of specific pathogen-associated molecular patterns recognized by proteins of the innate immune system is an essential foundation for development of therapeutic strategies that either directly target viral pathogens or enhance the host cell response to them.
Table of Contents
ABSTRACT. iv
ACKNOWLEDGEMENTS. vi
CHAPTER 1. 1
Introduction. 1
BACKGROUND. 2
RESEARCH GOALS. 14
REFERENCES. 17
CHAPTER 2. 31
Adenovirus VA RNA: An essential pro-viral non-coding RNA. 31
ABSTRACT. 32
INTRODUCTION. 33
Expression, Localization, and Structure of AdV VA RNAs. 35
Cellular Localization of VA RNAI. 39
VA RNA Structure. 41
VA RNAs Usurp Multiple Cellular Functions. 46
VA RNA interference with RNAi machinery. 46
Competition for nuclear export by Exportin 5 (Exp 5). 48
Dicer Saturation. 48
mivaRNA incorporation into RISC: a mechanism of viral control of host cell gene expression?. 50
VA RNAI-mediated inhibition of PKR. 54
VA RNAI regulation of 2'-5' oligoadenylate synthetase-1 (OAS1). 61
Pro-viral roles of Apical Stem-Central Domain fragment from Dicer-processing of VA RNAI. 65
VA RNAI induces type-I interferon late in infection. 68
Considerations for Ad vectors. 69
CONCLUSIONS. 70
ACKNOWLEDGEMENTS. 71
REFERENCES. 71
CHAPTER 3. 91
Plasmid template design and in vitro transcription of short RNAs within a 'structure cassette' for structure probing experiments. 91
ABSTRACT. 92
INTRODUCTION. 93
MATERIALS. 97
Generating a structure cassette plasmid containing a target RNA sequence. 97
Cloning Method 1: PCR. 98
Cloning Method 2: Direct ligation of chemically synthesized DNA oligonucleotides. 99
Analysis of transcribed RNA. 99
METHODS. 100
Generating structure cassette plasmids with new target RNAs. 100
Cloning Method 1: PCR. 102
Cloning Method 2: Direct ligation of chemically synthesized DNA oligonucleotides. 103
Analysis and preparation of transcribed RNA for probing experiments. 104
NOTES. 106
ACKNOWLEDGEMENTS. 110
REFERENCES. 110
CHAPTER 4. 112
Dissection of the adenoviral VA RNAI Central Domain structure reveals minimal requirements for RNA-mediated inhibition of PKR. 112
ABSTRACT. 113
INTRODUCTION. 113
EXPERIMENTAL PROCEDURES. 116
RESULTS AND DISCUSSION. 121
REFERENCES. 146
ACKNOWLEDGEMENTS. 151
FUNDING. 151
CHAPTER 5. 152
A novel RNA molecular signature for activation of 2'-5' oligoadenylate synthetase-1. 152
ABSTRACT. 153
INTRODUCTION. 153
MATERIAL AND METHODS. 155
RESULTS. 159
DISCUSSION. 173
ACKNOWLEDGEMENT. 177
FUNDING. 177
SUPPLEMENTAL FIGURES. 178
REFERENCES. 183
CHAPTER 6. 189
Conclusions. 189
VA RNAI INHIBITION OF PKR: OPEN QUESTIONS. 190
DICER PROCESSING OF VA RNAI. 194
FUNCTIONAL IMPLICATIONS OF THE 3'-ssPy MOTIF. 196
ADDITIONAL POTENTIAL PROVIRAL ROLES FOR VA RNAI. 202
THE FUNCTIONS OF EBV EBER-1 REMAIN UNCLEAR. 203
CONCLUSION. 204
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